culturing microorganisms 1

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Culturing Microorganisms

M 265 General Microbiology

Lab

Yasmine Al- shboul


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In nature microorganisms exist as mixed population of many different types.

However our knowledge of microbiology has come through study of isolated

species, grown in environments free from contamination by other livingforms.

Like all other living forms, microorganisms need suitable nutrients andenvironments.The culture medium must contain essential nutrients for thegrowth of a microbial culture ,and it must provide suitable surroundings for

growth : the proper Ph, osmotic pressure, atmospheric oxygen, and otherfactors.


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Nutritional Materials in Culture Media:

Water.

Carbon Source: Usually glucose.

Nitrogen Source: Either inorganic or organic e.g. beef extract orproteins.

Buffer System: e.g. carbonate or phosphate buffer.

Minerals: Required in small amounts e.g. iron, sulfur, phosphorus,etc

.Types of Media

According to composition:

Chemically Defined (Synthetic): Exact chemical compositionis known e.g. Minimal Salt Medium.

Complex (Undefined) Medium: Contains complex materials ofbiological origin such as Blood, Milk, Yeast, or Beef Extract.


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Synthetic media


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According to Agar (Solidifying Agent)Content:

Agar: Hydrocolloid derived from Red Algae.

Physical Properties of Agar: Melts at 100 C and remainsliquid until cooled to 40oC, it can't be metabolized bymost bacteria.

Liquid Media (Broth): Used for growth of pure cultures

e.g. Tripticase Soy Broth (TSB), Nutrient Broth (NB)

Semi-Solid Media:


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Agar agar, popular in Asia, is a gelatinoussubstance derived from red algae .


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Solid Media Forms:

Deep Agar Position: Used for bacterial

storage & for studying the gaseousrequirements of organisms.

Slanted Agar Position: Used for a variety ofpurposes.

Agar Plate Media (Petri Dishes): Largesurface area, used for isolation, studying ofpure cultures and for subculture.


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Deep solid agar in tube Slant agar


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According to Purpose:

All Purpose Media (Simple): Supports growth of most organisms e.g. NA or NB.

Enriched Medium: A base medium + special supplements such as Blood,Vitamins, and Amino Acids e.g. Blood Agar (BA) and Chocolate Agar (CA).

Differential Medium: More than one organism can grow, but these organisms canbe differentiated from each other based on color change or other criteria e.g.Blood Agar, KIA.

Selective Medium: Only allows one or more species to grow and suppressesothers by incorporating dyes, antibiotics, bile salts, or by adjusting PH orTemperature e.g. SS Agar.

Selective differential Medium: Inhibits growth of unwanted bacteria anddifferentiates between them e.g. MacConkey Agar (MA), Manitol Salt Agar (MSA).

Enrichment-Differential Medium: e.g. Blood Agar: (Alpha), (Beta), (Gamma)-Hemolytic.


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Chocolate Agar Blood agar


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Mannitol salt agar


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MacConkey Agar


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SS Agar


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Media Preparation:

Broth Media:

Weigh dehydrated media and dissolve in a specified amount of

water. Boil until clear.

Dispense in test tubes.

Close test tubes.

Autoclave at 121oC under 15lbs pressure for 15Min.

Agar Media:

Weigh dehydrated media and dissolve in a specified amount of water.

Boil until clear.


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For Test tubes:

Dispense in test tubes 1/3 or 3/4 full

Close test tubes

Autoclave at 121C under 15 lbs pressure for 15 Min. Allow test tube to cool at the appropriate position

For Petri Dishes:

Close flask with cotton and aluminum foil to prevent

vaporization.

Autoclave at 121oC under 15lbs pressure for 15Min.

Cool to 50oC.

Remove cap, Flame the mouth, and pour the liquid agar into

sterile Petri Dishes. Cover the Petri Dishes and allow cooling


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Heat Labile (Sensitive) compounds

that are to be added to the medium should be sterilized by filtration and

then added to the medium at 50oC.

Methods of Sterilization:

Sterile = Free from viable organisms.

Substances are either Sterile or Non-Sterile.

Sterilization: is the killing or removal of viable organisms byphysical of chemical methods.


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Chemical Sterilization Methods

Alcohol 70% Antiseptic and Disinfectant.

Formalin (Formaldehyde): Disinfectant. Chlorine (Sodium Hypochlorite): Disinfectant.

Ethylene Oxide Gas: used for contaminated medical tools.

Physical Sterilization Methods:

1. Heat:

The common and most important method used.

Sterilization requires consideration of temperature, and duration.

We should know the heat susceptibility of an organism to know what heat

treatment method to apply. Most


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Incineration: used for disposable material e.gneedles, swabs, gauze.

Dry Heat (Hot Air Oven): 160-180oC 1-3 Hours,

used for glassware, metal tools, and objects thatwon't melt.

Autoclaving (Steam under pressure or pressurecooker): 121oC/15lbs/15min. Multipurpose. Heatlabile substances will be denatured and destroyed.

Boiling: 100oC for 30min. Kills everything exceptsome spore forming bacteria.

Moist Heat (Tyndalization): Denatures andcoagulates proteins, kills vegetative bacteria, yeastand mold.

Pasteurization: Kills vegetative forms wilepreserving the flavor of foods.


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Filtration: The filter has a pore size smaller than bacteria. Used

for heat labile materials such as sugars and urea. e.g.Millipore filter, cellulose acetate disk.

Irradiation

(Microwave, UV, X-ray): destroysmicroorganisms, many organisms responsible for foodstorage are easily killed by irradiation.

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